LIBRARY Michigan State University This is to certify that the thesis entitled Use of Silver Zeolite As An Antimicrobial Agent in Packaging Film presented by Renae F. McKinley has been accepted towards fulfillment of the requirements for the Master degree in Packaging .7646“ (rs/£45 Major Professor‘s Signature A: 2mg V Date MSU is an Aflinnetive Action/Equal Opportunity Institution i PLACE IN RETURN BOX to remove this checkout from your record. ‘ To AVOID FINES return on or before date due. ! MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE NOV 1 3 2005 ‘A 11" .3 353.135 U FEB 217nm J2” .07 MAR 2 8 2038 108 r8 6/01 c:/ClRC/DateDue.p65-p. 15 USE OF SILVER ZEOLITE AS AN ANTIMICROBIAL AGENT IN PACKAGING FILM By Renae F. McKinley A THESIS Submitted to Michigan State University In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Packaging 2003 ABSTRACT USE OF SILVER ZEOLITE AS AN ANTIMICROBIAL AGENT IN PACKAGING FILM By Renae F. McKinley Antimicrobial properties of a commercially processed polyethylene film coated with AgIONTM silver zeolite were tested for its ability to inhibit growth of four strains of Listeria monocytogenes (CWD 95 and 246 from silage, CWD 201 from raw milk, and CWD 1503 from ground turkey). The surface of the films was inoculated with Listeria monocytogenes, and was inhibited 1.34 logs, respectively, on trypticase soy agar (TSA) containing 0.6%yeast extract (TSA-YE) after 24 hours of storage at 37°C. The antimicrobial properties of the film were also tested over 48 hours of storage at 4°C and 12°C, on beef bologna packaged in film inoculated with Listeria monocytogenes at a level of 106 CFU/cmz. The bologna was examined at specified intervals for numbers of Listeria monocytogenes, mesophilic aerobic bacteria (MAB), and lactic acid bacteria (LAB). During 48 hours of storage, Listeria populations decreased 0.39 and 0.05 logs, at 4°C and 12°C, respectively. At both temperatures, MAB populations decreased 0.07 logs at 4°C and 0.08 logs at 12°C using antimicrobial film compared to antimicrobial-free control film. At 4°C LAB populations were unchanged at 6.91 logs, while at 12°C the antimicrobial film decreased LAB populations 0.25 logs compared to antimicrobial-free film. To my family, BC, and Keisha iii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my research committee members, Dr. Susan Selke, Dr. Bruce Harte, and Dr. Elliot Ryser, for all their support and guidance. I am grateful to Dr. Elliot Ryser for allowing me to use his laboratory equipment and lending his expertise to complete this project. I express my most heartfelt thanks to my advisor, Dr. Susan Selke for her thoughtful advice and guidance throughout my graduate experience. I would like to also thank the School of Packaging (SOP) and the Center for Food and Pharmaceutical Packaging Research (CFPPR) for their financial support for this research. I owe a great deal of gratitude to Kim Lymn, Sheryl Brunken, Paul Tomianen, and Paul Engler from General Mills Packaging Research and Development for their financial and technical support throughout my research. I appreciate Dr. Richard Podhajny at Colorcon and Linda Foster at AgION for their technical support and providing me with film samples to complete this project. I owe a great deal of gratitude to Paweena Limjaroen, Lindsey Keskinen, and Arzu Cagri for their support and laboratory expertise throughout the course of my research. I would also like to thank my colleagues in Dr. Ryser’s laboratory and in the SOP. I sincerely appreciate Fernando Cardoso at the Michigan State University Statistics Consulting Center for his expertise in data analysis. My deepest appreciation goes to my mother, Jackie McKinley, my sister, Kristie McKinley, my family, my friends Sheryl Brunken, LaKeisha Hill, and Michelle Ryan for all their support and encomagement while completing this work. I am grateful for my extended Lansing family, Gladys and Hugh Scott, Connie and Karl Wilson, Jada Wilson, iv Elica Moss, and Kerri L. Harris. Thank you for all your thoughts, prayers, encouragement, and support during my matriculation at Michigan State University and my stay in East Lansing. I would especially like to thank Harrison Biles IV, my best friend, for his words of unrelentless guidance, dedication, encouragement, and understanding throughout my entire educational experience. TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES .................................................................. , ....................................... i x ABBREVIATIONS ........................................................................................................... x INTRODUCTION ............................................................................................................. 1 CHAPTER 1 ...................................................................... '. ............................................... 3 LITERATURE REVIEW ................................................................................................... 3 1.1. Antimicrobial Film .................................................................................................. 3 1.1.1. Antimicrobial Packaging Films ........................................................................ 3 1.1.2. Antimicrobial Edible Films ............................................................................... 4 1.2. Antimicrobial Additives .......................................................................................... 4 1.2.1. Organic Acids ................................................................................................... 5 1.2.1.1. Benzoic acid or sodium benzoate .............................................................. 5 1.2.1.2. Sorbic acid and sorbates ............................................................................. 6 1.2.2. Bacteriocins ....................................................................................................... 7 1.2.2.1. Nisin ........................................................................................................... 7 1.2.3. Parabens ............................................................................................................ 8 1.2.4. Natural Antrmrcrobrals ............................... 9 1.2.4.1. Lactoferrin .................................................................................................. 9 1.2.5. Curing Agents ................................................................................................... 9 1.2.5.1. Sodium Chloride ........................................................................................ 9 1.2.6. Metal ............................................................................................................... 10 1.2.6.1. Silver Ions ................................................................................................ 10 1.3. Listeria monocytogenes and Listeriosis ................................................................. 16 CHAPTER 2 ANTIMICROBIAL PROPERTIES OF SILVER ZEOLITE-COATED POLYETHYLENE PACKAGING FILM ........................................................................ 18 2.1. MATERIALS AND METHODS ........................................................................... 19 2.1.1. Film Preparation .............................................................................................. 19 2.1.2. Bacterial Strains .............................................................................................. 19 2.1.3. Film Inoculation and Storage .......................................................................... 19 2.1.4. Antimicrobial Properties ................................................................................. 20 2.1.5. Statistical Analysis .......................................................................................... 20 vi 2.2. RESULTS AND DISCUSSION ............................................................................ 20 2.3. CONCLUSION ...................................................................................................... 23 CHAPTER 3 INHIBITION OF LIST ERLA MONOCYTOGENES ON BEEF BOLOGNA USING SILVER ZEOLITE-COATED POLYETHEYLENE PACKAGING FILM .................... 24 3.1. MATERIALS AND METHODS ........................................................................... 25 3.1.1. Product ............................................................................................................ 25 3.1.2. Film Preparation .............................................................................................. 25 3.1.3. Bacterial Strains .............................................................................................. 25 3.1.4. Product Inoculation and Storage ..................................................................... 26 3.1.5. Microbiological Analysis ................................................................................ 26 3.1.6. Statistical Analysis .......................................................................................... 26 3.2. RESULTS AND DISCUSSION ............................................................................ 27 3.2.1. Listeria monocytogenes .................................................................................. 27 3.2.2. Mesophilic Aerobic Bacteria .......................................................................... 27 3.2.3. Lactic Acid Bacteria. ....................................................................................... 27 3.3. CONCLUSION ...................................................................................................... 28 CONCLUSIONS ............................................................................................................... 34 APPENDD( I .................................................................................................................... 36 APPENDIX 11 ................................................................................................................... 38 APPENDD( III .................................................................................................................. 47 APPENDIX IV .................................................................................................................. 49 BIBLIOGRAPHY ............................................................................................................. 51 vii LIST OF TABLES Table 2.1. Population of L. monocytogenes after 24 hours storage at 37°C. .................. 21 Table 3.1. Inhibition of Listeria monocytogenes, lactic acid bacteria, and mesophilic bacteria on bologna slices using control and antimicrobial films at 4°C .......................... 29 Table 3.2. Inhibition of Listeria monocytogenes, lactic acid bacteria, and meSOphilic bacteria on bologna slices using control and antimicrobial films at 12°C ........................ 30 viii LIST OF FIGURES Figure 1.1. Illustration of ion exchange in the formation of silver zeolite. ..................... 13 Figure 1.2. Illustration of silver ion release from film ..................................................... 15 Figure 2.1. TSA-YE plates from control film (top) and antimicrobial film (bottom) after 24 hours storage at 37°C. ................................................................................................................................. 22 Figure 3.1. Inhibition of L. monocytogenes on bologna samples wrapped in control and antimicrobial film during storage 4°C and 12°C. ............................................................. 31 Figure 3.2. Inhibition of mesophilic aerobic bacteria on bologna samples wrapped in control and antimicrobial film during storage at 4°C and 12°C. ...................................... 32 Figure 3.3. Inhibition of lactic acid bacteria on bologna samples wrapped in control and antimicrobial film during storage at 4°C and 12°C. ......................................................... 33 AN OVA ASTM CDC CF R CFU FSIS GRAS LAB MOX PBS PVDC SAS TSA TSA-YE TSB TSB-YE USDA ABBREVIATIONS Analysis of variance American Society for Testing and Materials Center for Disease Control Code of Federal Regulations Colony forming units Food and Drug Administration Food Safety Inspection Service Generally Recognized As Safe Lactic acid bacteria Mesophilic aerobic bacteria Modified Oxford Agar Butterfield’s phosphate buffer solution Polyvinylidene chloride Statistical Analysis System Rotations per minute Triptycase soy agar Triptycase soy agar + yeast extract Triptycase soy broth Triptycase soy broth + yeast extract United States Department of Agriculture University of Vermont Broth INTRODUCTION With the growing demand for convenience food products, today’s consumers are demanding food products with fewer or no preservatives. While meeting the demands of food consumers is important, an even greater concern in the food industry is post- processing food contamination of ready-to-eat food products from food-home microorganisms. The processed meat industry is constantly threatened by Listeria monocytogenes. Outbreaks of listeriosis (which is caused by Listeria monocytogenes) are major contributors to Class I microbial related food recalls. There have been several cases of listeriosis from consumption of processed meat that have resulted in fatalities. The Food and Drug Administration (FDA) estimates that Listeria monocytogenes causes estimated 2,500 cases of listeriosis and 500 deaths each year in the United States (FDA, 2001). In December 2000, an outbreak involving 29 cases in 10 states prompted the recall of approximately 14.5 million pounds of turkey and chicken deli meat due to probable Listeria monocytogenes contamination, which resulted in 7 fatalities (CDC, 2000). In an effort to address this growing concern, there has been enhanced interest during the past few years in the development of films containing antimicrobial additives. Siragusa, Cutter, and Willett incorporated bacteriocin in polyethylene to inhibit bacterial growth on beef carcass surface tissue sections (Siragusa et a1, 1999). Devlieghere et a1 (2000) used hexamethylenetetramine incorporated in low-density polyethylene to extend the shelf life of cooked ham. Labuza and Breene (1989) define antimicrobial film as a type of active packaging in which undesirable microorganisms in food are eliminated due to incorporation of antimicrobial additives into or coating of them onto food packaging materials. Because of the ability of such films to inhibit undesirable microorganisms, their availability for direct food contact use is increasing. These films also have the ability to potentially increase shelf life of food products. Colorcon’s No-Tox Products Division produces specialty silver zeolite antimicrobial inks and coatings for pharmaceutical, medical, and food packaging industries. The silver zeolite coatings, called AgIONm, are compliant with Food and Drug Administration (FDA) regulations for direct food contact applications for silver. AgIONTM has been found to inhibit growth and migration of bacteria, yeast, fungus and mold, while maintaining package performance (Podhajny, 2002). While the use of AgIONTM has proven beneficial in inhibiting surface contamination, there has been no published information regarding its effectiveness when in direct contact with food products and at differing storage temperatures. The hypothesis of this study was that polyethylene film coated with AgIONTM will inhibit the growth of Listeria monocytogenes both on the surface of the film and when in direct contact with sliced beef bologna stored at 4°C and 12°C. This study builds upon American Society for Testing and Materials (ASTM) E2180-01 and observations made by Colorcon. The objectives of this work are (1) to examine the effectiveness of polyethylene film coated with AgIONTM to inhibit the grth of four strains of Listeria monocytogenes (CWD 95 and 246 from silage, CWD 201 from raw milk, and CWD 1503 from ground turkey), and (2) to assess the coated film’s ability to retain its antimicrobial 1 properties while in direct contact with beef bologna slices stored at 4°C and 12°C. CHAPTER 1 LITERATURE REVIEW 1.1. Antimicrobial Film Antimicrobial film is a new and effective type of active packaging that inhibits undesired growth of microorganisms in food products. These films also have the ability to potentially increase product shelf life and enhance food safety. The use of antimicrobial additives is necessary in order for an antimicrobial film to be effective. Antimicrobial additives are materials used in various forms of packaging to reduce or prevent microbial growth (Han, 2000). i There are a variety of compounds that have been proposed as potential antimicrobial additives. Some of the common antimicrobial additives used to preserve food products include bacteriocins, organic acids and organic acid salts, parabens, natural antimicrobials, and curing agents. Whichever of these antimicrobial additives is used for food packaging, safety must be ensured. Typically, all antimicrobial agents used in film are approved by the Food and Drug Administration (FDA) in accordance with the Code of Federal Regulations (CF R) (Brody et a1, 2001). 1.1.1. Antimicrobial Packaging Films Antimicrobial films may be categorized as antimicrobial packaging films or antimicrobial edible films. The antimicrobial additives used for both types of antimicrobial films can be developed in two ways. The first is directly incorporating the antimicrobial additive(s) into the film through extrusion or extrusion coating. The second is to the coat the surface of the film with the antimicrobial additive(s). While each of these methods have advantages and disadvantages, both categories have proven effective in killing target organisms (Ming et a1, 1997;Siragusa et a1, 1999; Devlieghere et a1, 2000). Antimicrobial packaging films are non-edible films that serve as protection for a food product. Through the development of antimicrobial polyvinylidene chloride (PVDC) coploymer packaging films containing nisin, potassium sorbate, and sorbic acid, Limjaroen (2002) was able to inhibit the growth of four strains of Listeria monocytogenes using a diffusion assay. Limjaroen also determined that PVDC copolymer film containing 1.5% and 3.0% (w/v) sorbic acid decreased populations of L. monocytogenes 4.0-7.0 logs on beef bologna slices after 28 days of refi'igerated storage at 4°C. 1.1.2. Antimicrobial Edible Films Antimicrobial edible films can be prepared from proteins, polysaccharides, and/or proteins and lipids. They can be used in a variety of food packaging applications such as casings for hot dogs and sausages. Cagri et a1 (2002) determined that whey protein isolate edible films containing sorbic acid or p-aminobenzoic acid inhibited growth of Listeria monocytogenes, Escherichia coli 0157:H7, and Salmonella Ophimurium DT104 on trypticase soy agar (TSA). Cagri also determined that 0.5% to 1.0% sorbic acid or p- aminobenzoic acid was effective in decreasing populations of Listeria monocytogenes, Escherichia coli 0157:H7, and Salmonella typhimurium DT104 on beef bologna slices after 21 days storage at 4°C by 3.4-4.1 logs, 3.1—3.6 logs, and 3.1-4.1 logs, respectively. 1.2. Antimicrobial Additives In order for an antimicrobial film to be effective, an antimicrobial additive can be incorporated into or coated onto a packaging material. A variety of antimicrobial additives can be utilized to extend product shelf life. Some food components are naturally present or added during food formulation and processing and contain antimicrobial activity, thus contributing to food preservation. Of these antimicrobial additives, some are applied mainly to control microflora, while others have dual or multiple flmctions (Lou and Yousef, 1999). Some of the common antimicrobial additives used to preserve food products include organic acids such as benzoic, sorbic, and propionic acid; bacteriocins such as nisin; parabens; natlual antimicrobials such as lactoferrin; curing agents such as sodium chloride; and metals such as silver ions. Each will be discussed individually in terms of their possible use as antimicrobial additives in antimicrobial packaging films. 1.2.1. Organic Acids Most organic acids permitted in food are typically applied as acidulants (e. g. acetic and lactic acids), while their salt forms are used as preservatives (e.g. potassium sorbate and sodium benzoate). The effectiveness of these organic acids as antimicrobial agents is related to the amount of the undissociated form of the organic acid, which is related to pH of the medium and pKa of the acid (Lou and Yousef, 1999). Undissociated organic acids can pass through the cell membrane, dissociate inside the cytoplasm and interfere with the metabolic processes in the microbial cell. The antimicrobial activity of these acids can be attributed to cytoplasm acidification, and the antimicrobial effect of the particular anionic species (Lou and Yousef, 1999). 1.2.1.1. Benzoic acid or sodium benzoate In 1875, H. Fleck was the first to describe the preservative action of benzoic acid (Cagri, 2002). Sodium benzoate is the first chemical preservative permitted in food by the FDA. Both benzoic acid and sodium benzoate are Generally Recognized As Safe (GRAS) preservatives. Benzoic acid acts essentially as a mold and yeast inhibitor, but studies have shown that it can also inhibit Listeria monocytogenes using tryptose broth after 60 days storage (El-Shenawy and Marth, 1988). The antimicrobial activity of benzoate is related to pH. It is most effective in its undissociated form, with 60% of the compound undissociated at pH 4.0. Benzoic acid reduces the intracellular pH and also induces change in the morphology and appearance of microbial cells. Benzoic acid can also alter cell membrane function by producing pores that interfere with substrate uptake and electron transport. Sodium benzoate in combination with organic acids has proven effective in decreasing the microbial population of raw chicken. Hwang and Bechaut (1995) showed that raw chicken inoculated with Listeria monocytogenes, Salmonella, Campylobacter jejuni, Staphylococcus aureus, and Escherichia coli 0157 :H7 decreased in population by 1.0 log when immersed in a solution of 0.05% sodium benzoate/0.5% lactic acid (pH 2.64) for 30 minutes. 1.2.1.2. Sorbic acid and sorbates Sorbic acid is a straight chain a, B—unsaturated monocarboxylic acid (Sofos, 1989), and is classified as a GRAS preservative. The typical usage level is 0.02% to 0.3% in commercial foods. These concentrations are added to bakery, fruit, dairy, and meat products to inhibit yeasts, bacteria, and molds (Lueck 1980; Sofos and Busta, 1993; Kasrazadeh and Genigeorgis, 1995). Potassium sorbate is a commonly used salt of sorbic acid, and is highly soluble in water. Sorbic acid induces changes in the morphology and appearance of microbial cells. Its antimicrobial activity is generally enhanced at low temperatures. El-Shenawy and Marth (1988) showed that Y. enterocolitica, Pseudomonas putida, L. monocytogenes, and A. hydrophila were more sensitive to potassium sorbate at refrigerated temperatures than at higher temperatures. 1.2.2. Bacteriocins Bacteriocins are antimicrobial substances that encompass a peptide or protein component that is essential for their activity. With the exception of nisin, most bacteriocins have a narrow spectrum of inhibition, and only inhibit closely related species. Nisin exhibits a broad spectrum of inhibition against gram-negative bacteria. 1.2.2.1. Nisin Nisin, a polypeptide that is synthesized by Lactococcus lactis subspecies lactis, is one of the most widely studied bacteriocins. In 1998, the FDA approved nisin for pasteurized processed cheese with no more than 250 parts per million (ppm) nisin in the finished product (Lou and Yousef, 1999). Being a natural additive and having the status of GRAS contributes to its wide use (Bower et a1, 1995). Composed of 34 amino acids (Jung, 1991), nisin possess amphiphilic characteristics, with clusterslof hydrophobic and hydrophilic residues at the N- and C-terminus. At the N— and C-terminus Cross and Morell (1971) identified didehydroalanyllysine and isoleucine. The highly reactive double bonds and thioether cross-linkages are suggested to be responsible for important properties of the nisin molecule, such as thermostability, acid tolerance, and bactericidal. The most significant application of nisin may be in the inhibition of gram-positive bacteria, such as Listeria monocytogenes and Clostridium spp. (Ming et al, 1997, Bower et a1, 1995, Padgett et al, 1998). However, when combined with chelating agents, it has been suggested that nisin may be effective against gram-negative bacteria (Wells et al, 1998; Boziaris and Adams, 1999). Liu and Hansen (1990) determined that the biological activity of nisin decreased at high pH values. The stability, solubility, and biological stability are pH dependent, and nisin becomes insoluble at alkaline and neutral conditions. Nisin has been effective in cheese products such as cheese spreads and cold-pack cheese (Delves-Broughton, 1990). It is effective against bacteria in meat and meat products. Bell and Dlacy (1986) reported that nisin inhibited growth of Bacillus licheniformis in fermented and cured meat products. 1.2.3. Parabens Parabens are esters of p-hydroxybenzoic acids, which are made by esterification of the carboxyl group of benzoic acid. Due to their ability to remain undissociated at pH values of up to 8.5, most parabens are active at pH values of 3.0 to 8.0. Of these esters, methyl, propyl, and heptyl parabens are approved in some countries for direct food use (Lou and Yousef, 1999). While parabens can be effective in a wide range of foods, they are mostly used in butter, margarine, meat products, and maple syrup (Limjaroen, 2002). Parabens are more effective in the inhibition of gram-positive bacteria than of gram-negative bacteria, due to their decreased polarity. Parabens are generally more effective against mold and yeasts than against bacteria. Concentrations of 32 to 1000 jig/ml are normally needed to inhibit bacteria and fungi using esters of p-hydroxybenzoic acid in laboratory media after 24 and 48 hours storage (Kato and Shibasaki, 1975; Jermini and Schmidt-Lorenz, 1987; Thompson, 1991; Juneja and Davidson, 1993). Since parabens are phenolic derivatives, their antimicrobial mechanisms are similar to those of phenols and phenol-related compounds. Judis (1963) proposed that phenol physically damages the cytoplasmic membrane of microorganisms, resulting in the release of cytoplasmic compounds. Eklund (1980) showed that parabens inhibit nutrient uptake through the cytoplasmic membrane. This results in inhibition of membrane transport and the electron transport system. 1.2.4. Natural Antimicrobials 1.2.4.1. Lactoferrin A glycoprotein found in mammalian milk, lactoferrin exerts its antimicrobial activity through the binding of iron, and can bind two iron atoms per molecule (Lou and Yousef, 1999). This protein has proven to be effective against Micrococcus spp., Bacillus subtilis, Listeria monocytogenes, Bacillus sterothermophilus, and Escherichia coli (Orarn and Reiter, 1968; Reiter, 1978; Payne et al, 1990). It was reported that lactoferrin causes cell death by chelation of iron, calcium, and magnesium ions (Ashton and Busta, 1968). Listeria monocytogenes was inhibited by lactoferrin, which was directly related to iron availability in the medium, with Listeria monocytogenes surviving best in an iron-rich media (Payne et al, 1989). However, it was reported that lactoferrin inhibited many bacteria in an iron-rich environment. For Escherichia coli, lactoferrin chelated cations that stabilized lipopolysaccharldes, which led to increased permeability of the outer membrane to hydrophobic compounds (Arnold et a1, 1982). 1.2.5. Curing Agents 1.2.5.1. Sodium Chloride Sodium chloride is a food preservative used since ancient times. It can be used alone or in combination with other food preservation techniques (e.g. fermentation or pasteurization). Most bacterial food-home pathogens are susceptible to sodium chloride, with the exception of Staphylococcus aureus, which grows at a low water activity (0.83 to 0.86) (McLean et a1, 1968). Listeria monocytogenes can grow at concentrations of up to 10% sodium chloride. Sodium chloride exerts its antimicrobial activity through its ability to reduce water activity in food. Microbial cells lose water when the water activity of the external environment is reduced, which results in growth inhibition or possible death (Sperber, 1983). Sodium chloride also alters pH, limits oxygen solubility, and is toxic for microbial cells (Banward, 1979). 1.2.6. Metal 1.2.6.1. Silver Ions Silver ions have the strongest antimicrobial activity among other metallic ions (Brody et a1, 2001). When compared to other metallic ions metallic silver does not release the ion easily, thus making its antimicrobial activity not as strong in its metallic state. Silver ions are naturally occurring and effective microbe inhibitors. The use of silver zeolite as an antimicrobial additive is a new approach to prevent microbial growth in food products. Silver is safe and relatively inert, making it safe for direct human contact. FDA clearance allows for a maximum of 2% silver content by weight of the polymer (FDA, 2003). Human exposure to silver and silver compounds can occur orally, derrnally, and by inhalation. Ingestion, dermal absorption, or inhalation of silver nitrates my cause argyria. Argyria is a disease caused by long term exposure to silver nitrate resulting in a gray or blue-gray permanent discoloration of the skin. 10 Silver salts possess the greatest potential for antimicrobial activity (e.g. silver nitrate and Ag-zeolite) (Brody et a1, 2001). Silver nitrate that forms silver ions in water has strong antimicrobial activity. The antimicrobial activity of silver is achieved through an interference with metabolic functions of the respiratory and electron-transport systems of microorganisms. Silver ion is absorbed by the surface of the microbial cell through active transport, thus inhibiting a range of metabolic processes necessary for sustaining life (Brody et a], 2001). To facilitate antimicrobial activity, Ag—zeolite retains Ag+ ions in stable and effective conditions (Brody et a1, 2001). In order for Ag- zeolite to be formed, sodium ions in a‘zeolite are substituted with silver ions (Figure 1.1). Ag-zeolite is effective against bacteria, firngi, and yeast, but does not demonstrate antimicrobial activity against spores of heat-resistant bacteria (Brody et a1, 2001). Because of its expense, Ag-zeolite for packaging is usually laminated as a thin coextruded layer (3-6 um) (Brody et a1, 2001). To order to provide adequate heat-seal and other physical film properties, the normal incorporation level of Ag-zeolite is l to 3% (Brody et al, 2001). Silver ions are activated and released by moisture in the air, which allows for a steady rate to maintain the antimicrobial surface, thus ensuring product longevity and integrity. Slow release of Ag-zeolite is critical for optimal antimicrobial activity. Slow diffusion of an antimicrobial agent is important because it allows for longer and more effective antimicrobial action, which provides longer product shelf life (Brody et a1, 2001; Brody 2001). The No-Tox Products Divisions of Colorcon produces specialty antimicrobial silver zeolite coatings called AgIONTM for pharmaceutical, medical, and food packaging industries. AgIONTM coatings that are compliant with FDA regulations for direct food 11 contact applications use less than 0.001% of silver by. weight of dry coating (AgION, 2001). According to Colorcon, zeolite, ion exchange, and silver must be present in order for the AgIONTM to be effective (Cooksey, 2001) (Figure 1.1). 12 ‘l._ | -. Tm EXCHANGE : _- .i ‘. 4' -l ! .." ~_ _ Jae-r Baffin. ‘ - '.'_ ,_ 1'” "All , . . , ,.. - m- J. .n-z- I‘l, ‘, . r‘. ;- , r ’3 l s 7- . a v _, .1 . L' - r'. g .y, 1 r. r , ,r t if" " J ' I E? as!” chassis. in It? @. Figure 1.1. Illustration of ion exchange in the formation of silver zeolite. AgIONTM is dispersed as a fine solid in a proprietary water or solvent based coating. Solvent-based coatings were found to provide better release of the additive when tested against Listeria monocytogenes (Podhajny, 2002). The inorganic silver in zeolite carriers is antimicrobial on contact and technically feasible to fabricate, but the required migration from polymers is minimal (Brody, 2001). The coating itself is so effective that its shelf life is typically the shelf life of the product (Podhajny, 2002). Using such antimicrobial packaging materials is not a substitute for good sanitation and handling practices. These antimicrobial packaging materials should be used as an additional protective measure to help ensure the safety and high quality of food products (Cooksey, 2001). 14 Surface Film of Moisture Bacteria Silver ion release Environment Surface Layer No—Tox®AM Coating Silver Zeolite Figure 1.2. Illustration of silver ion release from film. 1.3. Listeria monocytogenas and Listeriosis Post-processing contamination of ready-to-eat meat products has become a growing concern in the processed meat industry. Listeria monocytogenes is continuously posing a threat to the meat industry as one of the major contributors to post-processing contamination. Listeria monocytogenes is a gram positive, psychrotropic, non-spore forming bacteria that is common in the environment. Listeria monocytogenes typically grows between 345°C, with an optimum temperature growth range of 30-37'C. This pathogen can grow at a pH range of 5.6-9.6, with an optimum range of slightly alkaline to neutral pH values (Donnelly et a1, 1992). Listeria monocytogenes has proven difficult to control in food processing facilities. Mafu et al (1990) demonstrated that Listeria monocytogenes can attach to and survive on rubber, stainless steel, glass, and polypropylene food contact surfaces. Listeria monocytogenes contamination can often occur in processed meat products such as bologna, deli meats, hot dogs, poultry, and meat spreads. Consumption of a food product contaminated with Listeria monocytogenes can cause listeriosis. Listeriosis is a food-bome illness that is a major contributor to Class I microbial related food recalls. It is estimated that 5,000 Americans die each year from food-bome illnesses (CDC, 2002). While anyone can be affected by listeriosis, it can be fatal for the elderly, newborns, the fetus of pregnant women, and the irnmunocompronrised (U SDA-F SIS, 2002). Those with listeriosis often develop meningitis, sepsis, or meningoencephalitis (Slutsker and Schuchat, 1999). Pregnant women with listeriosis typically develop mild flu-like symptoms, fever, headaches, or gastrointestinal symptoms (Slutsker and Schuchat, 1999). Some infections in pregnant 16 women may result in preterm labor, amnionitis, spontaneous abortion, or stillbirth (Slutsker and Schuchat, 1999). During the second half of 2000, an outbreak involving 29 cases in 10 states (including 7 fatalities) prompted the recall of approximately 14.5 million pounds of turkey and chicken deli meat due to probable Listeria monocytogenes contamination (CDC, 2000). ' The United States Department of Agriculture (USDA) and the Food Safety Inspection Service (F SIS) have been working diligently to develop methods to reduce the number of listeriosis outbreaks. On June 4, 2003, an interim final rule that requires federal establishments that produce ready-to-eat meat and poultry products to reduce incidences of Listeria monocytogenes. This rule requires those establishments that produce ready-to-eat products that are exposed to the environment after cooking to devise written programs such as Hazard Analysis and Critical Control Point (I-IACCP) systems, Sanitation Standard Operating Procedures (Sanitation SOPs), or other preliminary programs to control Listeria monocytogenes. Verification of the program’s effectiveness will be achieved through testing, and results will be shared with the FSIS. The establishments will also provide the FSIS with information on production volumes as well as any other information on the product affected by regulation (U SDA-FSIS, 2003). While the use of antimicrobial films does not replace this ruling, their implementation can assist with this effort. l7 CHAPTER 2 ANTIMICROBIAL PROPERTIES OF SILVER ZEOLITE-COATED POLYETHYLENE PACKAGING FILM l8 2.1. MATERIALS AND METHODS 2.1.1. Film Preparation Commercially processed antimicrobial and control films were aseptically cut (5.08 x 5.08 cm) and sterilized by UV light for 20 minutesiusing a Class II A/B3 Biological Safety Cabinet (Forma Scientific Inc., Marietta, OH). The film samples were then aseptically placed in 15-mm disposable polystyrene petri dishes. 2.1.2. Bacterial Strains Four strains of Listeria monocytogenes (CWD 95 and CWD 246 from silage CWD 201 from raw milk, and CWD 1503 from ground turkey) were obtained from CW. Donnelly (Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT). All strains were maintained at -70°C in trypticase soy broth (T SB) (Difco Laboratories, Detroit, MI) containing 10% (v/v) glycerol and subcultured twice in TSB containing 0.6% (w/v) yeast extract (TSB-YE) (Difco) at 35°C 18 to 24 hours before use. Following incubation, 2.5 ml of each culture was combined and centrifuged using a Sorvall Super T21at 10,000 rpm for 10 minutes at 4°C (Kendro Laboratory Products, Newtown, CT), and was resuspended in 10 ml sterile Butterfield’s phosphate buffer (PBS) (Sigma Chemical Company, St. Louis, MO) (neutralized to pH 7.2 with 1 N sodium hydroxide). One ml of the inoculum was added to 99 ml of sterile PBS (Sigma) (neutralized to pH 7.2 with l N sodium hydroxide). 2.1.3. Film Inoculation and Storage ’ Antimicrobial and control films were aseptically placed in 15-mm disposable polystyrene petri dishes and inoculated with 0.5 ml of the diluted culture. Immediately after the films were inoculated, stomacher bags (5.08 x 5.08 cm) were aseptically placed 19 on each film sample to maintain inoculum on the film. These petri dishes were covered and stored in plastic containers with 200 ml of boiled deionized water. The plastic containers were stored at 37°C. All experiments were replicated three times. 2.1.4. Antimicrobial Properties ‘ Samples were examined for numbers of Listeria immediately after inoculation and again following 24 hours of storage at 37°C. For analysis, the films were aseptically added to 20 m1 of sterile PBS (Signa) and homogenized using a Stomacher 400 (T ekmar Co., Cincinnati, OH) for 2 minutes, after which 0.1 ml of the dilution was surface plated on trypticase soy agar (TSA) containing 0.6% yeast extract (T SA-YE). Following 24 hours of incubation at 37°C, the number of colonies was counted. All experiments were replicated three times. 2.1.5. Statistical Analysis All experiments were performed in triplicate. Film type and storage time data was analyzed by two-way analysis of variance (ANOVA) using SAS Statistical Analysis System (SAS Institute Inc., 2001). Means were compared using Tukey—Kramer adjustment at p=0.05. 2.2. RESULTS AND DISCUSSION Control films without silver zeolite showed no antimicrobial activity against the L. monocytogenes during storage. However, the antimicrobial film showed antimicrobial activity against L. monocytogenes (T able 2.1 and Figure 2.1.). At the initial incubation of the film samples, there was no significant difference between the control and antimicrobial film (p <0.05). There was a significant difference between the control and 20 Table 2.1. Population of L. monocytogenes after 24 hours storage at 37°C. Storage Time (Hours) Film Types 0 ' 24 Silver Zeolite 5.23%: 0.21 <3.89°=|: 0.00 Control 533‘s 0.5 6.26%: 0.03 Unit=CFU/cm2 (logro) Geometric mean :l: standard deviation (n=3). Means with different superscripts are significantly difl’erent (p<0.05). 21 Figure 2.1. TSA-YE plates from control film (top) and antimicrobial film (bottom) after 24 hours storage at 37°C. 22 antimicrobial film after 24 hours (p<0.05) (Table 2.1). There was no growth observed in TSA-YE for the antimicrobial film (Figure 2.2). Therefore, the antimicrobial film used in this study proved effective in inhibiting the growth of L. monocytogenes in direct contact after 24 hours. A 2.3. CONCLUSION This study showed that coating silver zeolite coating on polyethylene led to the inhibition of L. monocytogenes; whereas the control film without antimicrobial coating was found to be non-inhibitory. Given the results of this work, it is likely that this film may prove useful for reducing post-processing contamination of to ready-to-eat meat products. 23 CHAPTER 3 INHIBITION OF LISTFRIA MONOCYTOGENES ON BEEF BOLOGNA USING SILVER ZEOLITE-COATED POLYETHEYLENE PACKAGING FILM 24 3.1. MATERIALS AND METHODS 3.1.1. Product Pre-sliced (~ 3 mm), commercially processed beef bologna (diameter 9.6 cm) was obtained from a local supermarket. As reported by the manufacturer, the all-beef bologna contained beef, salt, sodium lactate, flavor, dextrose, hydrolyzed beef stock, autolyzed yeast, sodium phosphate, sodium diacetate, sodium erythorbate, sodium nitrite, and extractives of paprika At the time of purchase, the beef bologna had a pH value of 6.0. The product used for all trials was from the same production lot. Enrichment analysis was conducted to confirm the absence of L. monocytogenes in the bologna prior to testing as outlined in the Compendium of Methods for the Microbiological Examination of Foods (Donnelly et al, 2001). 3.1.2. Film Preparation Commercially processed film samples were then aseptically cut (6.35 x 3.81 cm) and sterilized by UV light using a Class II A/B3 Biological Safety Cabinet (Forma Scientific Inc., Marietta, OH) for 20 minutes. The film was then aseptically placed in 15-mm disposable polystyrene petri dishes. 3.1.3. Bacterial Strains Four strains of Listeria monocytogenes (CWD 95 and CWD 246 fiom silage CWD 201 from raw milk, and CWD1503 from ground turkey) were acquired from CW. Donnelly (Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT). Each strain was maintained at -70°C in trypticase soy broth (T SB) (Difco Laboratories, Detroit, MI) containing 10% (v/v) glycerol and subcultured twice in TSB containing 0.6% (w/v) yeast extract (TSB-YE) (Difco) at 35°C 18 to 24 hours prior 25 to use. The four strains (2.5 ml each) of L. monocytogenes were combined and centrifuged (Sorvall Super T21, Kendro Laboratory Products, Newtown, CT) for 10 minutes at 4 °C at 10,000 rpm, and resuspended in 10 ml of sterile 0.1% (w/v) peptone water (Difco). I 3.1.4. Product Inoculation and Storage Antimicrobial and control films were inoculated with 0.1 ml of a diluted culture to obtain an inoculum level of log 6.0 (:l: 0.1) CFU/cmz. The inoculum was evenly spread on the film surface that was to be in contact with the bologna sammes using a sterile glass rod. Bologna slices were individually wrapped in the inoculated film samples and stacked 3 slices high in sterile 150-mm. diameter petri dishes. The bologna was then covered and stored aerobically at 4°C and 12°C. 3.1.5. Microbiological Analysis The slices of bologna were examined for level of inoculum, lactic acid bacteria, and meSOphilic aerobic bacteria immediately after inoculation, and again following 2, 4, 6, 8, 24, and 48 hours of storage at 4°C and 12°C. 10-g bologna slices were each added to 40 ml of sterile 0.1% (w/v) peptone water (Difco) and homogenized in a stomacher (Tekmar Co., Cincinnati, OH) for 3 minutes. Appropriate dilutions of peptone water (Difco) were plated on Modified Oxford Agar (MOX) (Difco), Plate Count Agar (Difco), and MRS Lactobacillus Agar (Difco) to quantify Listeria monocytogenes, mesophilic aerobic bacteria (MAB), and lactic acid bacteria (LAB). 3.1.6. Statistical Analysis All experiments were conducted in triplicate. Film type, storage time, and temperature data was analyzed by three-way analysis of variance (AN OVA) using SAS 26 Statistical Analysis System (SAS Institute Inc., 2001). Analysis of means was compared at a 95% confidence level (p=0.05) using Tukey-Klamer adjustment. 3.2. RESULTS AND DISCUSSION 3.2.1. Listeria monocytogeues Antimicrobial and control films were initially inoculated to contain 10° CFU/cm2 and examined after 48 hours storage at 4°C and 12°C. The population distributions of L. monocytogenes during 48 hours at the two temperatures are shown in Tables 3.1 and 3.2. The antimicrobial film decreased populations of L. monocytogenes by 0.39 logs, from 6.79 to 6.40 logs, for the bologna slices after 48 hours of storage at 4°C (F igurc 3.1). A similar effect was observed for bologna slices after 48 hours of storage at 12°C, with L. monogrtogenes decreasing by 0.05 logs, from 6.78 to 6.73 logs. After 48 hours storage at 4°C, Listeria counts increased 0.1 logs using film without silver zeolite antimicrobial coating, and 0.09 logs at 12°C. 3.2.2. Mesophilic Aerobic Bacteria Numbers of mesophilic aerobic bacteria (MAB) remained unchanged at about 6.91 logs for bologna slices stored 48 hours at 4°C with antimicrobial film (Figure 3.2). MAB populations increased by 0.07 logs, from 6.73 to 6.80 logs, with antimicrobial-free film samples. The MAB population, after 48 hours storage at 12°C, decreased by 0.25 log, from 6.90 to 7.15 logs with antimicrobial film, while MAB with antimicrobial-free film increased by 0.16 logs (Figure 3.2). 3.2.3. Lactic Acid Bacteria Using the antimicrobial film, LAB increased from 6.73 to 6.80 logs on the bologna slices after 48 hours storage at 4°C; a similar effect was observed with 27 antimicrobial-free fihn (Figure 3.3). After 48 hours storage at 12°C, LAB populations increased by 0.08 logs with the antimicrobial film, and antimicrobial-free film increased by 0. 16 logs. 3.3. CONCLUSION Polyethylene coated with silver zeolite reduced the growth of L. monocytogenes when in contact with bologna slices at 4°C and 12°C for 48 hours. The film shows little promise for extending the shelf life of temperature-abused sliced beef bologna due to low population decreases. 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N0. e e0+m0m m 0m .e 0e e 0030“. 00.00.00 00000 00000... 3.00.00 00000 00000... 3.00.00 00000 m0... 033:0 ow00o>< 000.00 m<2 00.02.0— 0m00o>< 000.00 0.280.: 00.320 ow000>< 000.00 000.... 0.800 0 2.0.0. 46 APPENDIX III 47 FILM PREPARATION Corona-treated polyethylene was aseptically cut (17.78 x 22.86 cm) and taped onto manila folders. Two ml of F GN-5415 silver zeolite coating was then coated onto the film with using a #3 Meyer rod (Colorcon, West Point, Pennsylvania). The coating was dried using a hot gun (Colorcon). 48 APPENDIX IV 49 ENRICHMENT PROCEDURE FOR ISOLATION OF L. MONOCYTOGENES Sample Preparation (from section 38.513 USDA’s Enrichment Procedure for Isolation of L. monocytogenw From Meat Products, Compendium of Methods for the Microbiological Examination of Foods, 1992) Surfaces of beef-bologna packages were swabbed with 3% hydrogen peroxide before opening. Bologna slices were cut into 25-g pieces on a sterilized aluminum baking pan using a sterilized knife and forceps. 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